As for identification of bacteria, for example, a method for identifying bacteria using DNA microarray technique has been developed, in addition to a conventionally-used identification method via culture and an identification method by staining or a method comprising measuring a substance associated with the bacterial metabolism such as ATP. Particularly, a technique comprising the steps of synthesizing as a probe nucleic acids having a nucleotide sequence which is specific to a gene sequence of certain bacteria, and fixing the probe on a substrate to allow the probe to hybridize with a gene amplified from a specimen is currently applied in various advanced ways. It is because this technique uses a nucleotide sequence specific to each bacterial species, and thus allows an accurate detection (see patent documents 1 to 3 and non-patent document 1).
One example of the quantitative-analysis method is a detection method by real-time PCR method. The real-time PCR method enables to perform a quantitative detection using trace amounts of sample, but the test takes time and in addition, the suitable number of cycles for quantification has to be found out for each template and primer for a highly-reproducible quantification, which results in a complicated operation.
Other method such as NASBA (Nucleic Acid Sequence Based Amplification) method which is for amplifying RNA-specific nucleic acids is proposed as a method capable of detecting a particular bacterial species or breed. NASBA method comprises amplifying an antisense RNA which is complementary to the intended RNA using two kinds of primers. Since with this method, it is possible to amplify nucleic acids in a short time using RNA as a template under the constant temperature of 41° C., the reaction can be performed at ordinary temperature without the need of a temperature-control device or the like. Further, the Tm value, etc. of the primer does not have to be taken into consideration, and this allows a simple detection of plural kinds of nucleic acids. Further, unlike RT-PCR, amplification and detection of RNA can be performed in the presence of DNA and thus possible false-positive cases due to dead bacteria can be eliminated by assessing viable bacteria. However, it has been said that while NASBA method is capable of detecting plural kinds of nucleic acids at the same time qualitatively and simply, a quantitative and simple detection is difficult with this method.
Meanwhile, pneumonia is in fourth place in the Japanese cause-specific death rates. It is frequently developed as a complication associated with an underlying disease such as cancer and is known as a disease affecting a very large number of individuals. Conventionally, a culture test which is performed as a search test for a microorganism that causes pneumonia (causative bacteria) takes at least several days and it takes nearly a week if a drug sensitivity test is further performed on the cultured causative bacteria. Therefore, this test method has not become a method which sufficiently contributes to the treatment selection. In a case of severe pneumonia which requires hospitalization in an intensive-care unit (ICU), a rapid and accurate determination of causative bacteria is very important in treatment selection, and it has been reported that an appropriate initial treatment definitely increases the life-saving rate of pneumonia patients. However, in reality, the actual state is that the treatment has to be carried out with the causative bacteria unidentified, because no techniques for identifying causative bacteria that may replace the culture method have been established yet. Consequently, use of antibiotics based on experience is unavoidable, which may potentially result in an appearance of drug-resistant bacteria.
Bacterial species that are frequently seen to cause pneumonia account for nearly 50% of the causative bacteria of pneumonia, and there are about 20 to 30 kinds of major causative bacteria including viruses. Some of these cannot be cultured by a common technique, and in many cases, it is difficult to determine the causative bacteria by culture method. Further, a conventional method for detecting microorganisms using DNA microarray technique was effective for detecting causative bacteria of pneumonia which cause pneumonia even with a very small infective dose, and for detecting plural kinds of bacteria at the same time, but such method had a problem in terms of a quantitative and highly-reproducible analysis. Particularly for pneumonia which needs to be treated by appropriately choosing an antibiotic depending on the bacterial amount, it is very important to detect plural kinds of causative bacteria of pneumonia at the same time and to quantitatively analyze the detected signals. Further, optimal therapeutic agents differ depending on the kind of the causative bacteria, but in actual state, it is unavoidable to start the treatment before determining the causative bacteria from the viewpoint of medical ethics. In order to solve these problems, the development of a technique capable of detecting specific bacteria rapidly and quantitatively from among plural bacterial species has been awaited.
Patent Document 1: Japanese Laid-Open Patent Application No. 2002-512688
Patent Document 2: Japanese Laid-Open Patent Application No. 2006-025791
Patent Document 3: Japanese Laid-Open Patent Application No. 2006-061155
Non-Patent Document 1: Schena M. et al. (1996) Proc. Natl. Acad. Sci. USA. 93(20): 10614-9